title: Expression and biochemical characterization of the human enzyme N-terminal asparagine amidohydrolase.
abstract: The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein, we report the bacterial expression, purification, and biochemical characterization of human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the Cys75Ala point mutation. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating L-Met is important for the function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway.
authors.affiliation: Department of Chemical Engineering, Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas, Austin, Texas 78712, United States.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Cantor
authors.forename: Everett M
authors.initials: EM
authors.lastname: Stone
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008956:Models, Chemical; D015394:Molecular Structure; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D008670:Metals; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; D000581:Amidohydrolases; C425491:N-terminal asparagine amidohydrolase; D003545:Cysteine